Central role of the ramAR locus in the multidrug resistance in ESBL-Enterobacterales

ABSTRACT The aim of this study was to evaluate the proportion of resistance to a temocillin, tigecycline, ciprofloxacin, and chloramphenicol phenotype called t2c2 that resulted from mutations within the ramAR locus among extended-spectrum β-lactamases-Enterobacterales (ESBL-E) isolated in three intensive care units for 3 years in a French university hospital. Two parallel approaches were performed on all 443 ESBL-E included: (i) the minimal inhibitory concentrations of temocillin, tigecycline, ciprofloxacin, and chloramphenicol were determined and (ii) the genomes obtained from the Illumina sequencing platform were analyzed to determine multilocus sequence types, resistomes, and diversity of several tetR-associated genes including ramAR operon. Among the 443 ESBL-E strains included, isolates of Escherichia coli (n = 194), Klebsiella pneumoniae (n = 122), and Enterobacter cloacae complex (Ecc) (n = 127) were found. Thirty-one ESBL-E strains (7%), 16 K. pneumoniae (13.1%), and 15 Ecc (11.8%) presented the t2c2 phenotype in addition to their ESBL profile, whereas no E. coli presented these resistances. The t2c2 phenotype was invariably reversible by the addition of Phe-Arg-β-naphthylamide, indicating a role of resistance-nodulation-division pumps in these observations. Mutations associated with the t2c2 phenotype were restricted to RamR, the ramAR intergenic region (IR), and AcrR. Mutations in RamR consisted of C- or N-terminal deletions and amino acid substitutions inside its DNA-binding domain or within key sites of protein–substrate interactions. The ramAR IR showed nucleotide substitutions involved in the RamR DNA-binding domain. This diversity of sequences suggested that RamR and the ramAR IR represent major genetic events for bacterial antimicrobial resistance. IMPORTANCE Morbimortality caused by infectious diseases is very high among patients hospitalized in intensive care units (ICUs). A part of these outcomes can be explained by antibiotic resistance, which delays the appropriate therapy. The transferable antibiotic resistance gene is a well-known mechanism to explain the high rate of multidrug resistance (MDR) bacteria in ICUs. This study describes the prevalence of chromosomal mutations, which led to additional antibiotic resistance among MDR bacteria. More than 12% of Klebsiella pneumoniae and Enterobacter cloacae complex strains presented mutations within the ramAR locus associated with a dysregulation of an efflux pump called AcrAB-TolC and a porin: OmpF. These dysregulations led to an increase in antibiotic output notably tigecycline, ciprofloxacin, and chloramphenicol associated with a decrease of input for beta-lactam, especially temocillin. Mutations within transcriptional regulators such as ramAR locus played a major role in antibiotic resistance dissemination and need to be further explored.

1st Editorial Decision Re: Spectrum03548-23 (Central role of the ramAR locus in the multidrug resistance in ESBL Enterobacterales) Dear Dr. Francois GRAVEY: Thank you for the privilege of reviewing your work, which has now been reviewed by 2 experts in your field.Both have suggested important revisions in order for your manuscript to be reconsidered for publication.Reviewer 1 has highlighted the need to include additional references and statements that dampen some of your claims, while Reviewer 2 has indicated the need for performing some additional experiments and re-analyzing the data.Inclusion of acrAB expression data, for instance, would support the claim about specific impacts of some mutations.There are also concerns about the presentation of the manuscript, which could be enhanced by additional rounds of edits and better organization of the different sections.Similarly, Reviewer 2 pointed out issues and inconsistencies with the supplementary data.Keeping these and the remaining comments in mind, we would like to invite you to submit a revision for consideration.The complete list of reviewer comments are included below.
If you choose to resubmit, then please be sure that your BioProject sequences are made publicly available prior to submission and return the manuscript within 60 days.If you cannot complete the modifications within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, then please notify me immediately so that the manuscript may be formally withdrawn from consideration by Spectrum.

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Reviewer #1 (Comments for the Author): Specific Comments concerning the manuscript intitled : « Central rôle of the RamAR locus in the multidrug resistance in ESBL Enterobacterales ».Spectrum03548-23

General comments :
The authors screened 443 ESBL Enterobacteria for efflux resistance phenotype to 4 antibiotics (temocillin, tigecycline, ciprofloxacin, chloramphenicol), and identified those with mutations in the ramAR locus.The RamA regulatory protein is known to be primarily involved in regulating the active efflux of antibiotics in Klebsiella, Salmonella and Enterobacter, by modulating expression of AcrAB-TolC pump.In this study, the combined efflux-dependent resistance phenotype for the four antibiotics was found only in K. pneumoniae and E. cloacae complex, but not in E. coli.Among these 2 groups, analysis of the sequences of the genes encoding all the regulatory proteins involved in efflux pump regulation, as well as the structural genes of the pump proteins, shows that most of the mutations found in MDR strains are identified at the level of ramR gene and the ramAR intergenic region.This is not a new finding in these bacteria, as previous studies have shown that RamAR plays a major role in the overexpression of AcrAB-TolC efflux pumps, and mutations in RamR have also been identified.However, none of the previous studies analyzed such a large number of isolates and identified so many different mutations at ramAR locus.This work reinforces the observations of a number of previous publications, and has the advantage of having performed a sequence analysis of recently isolated clinical strains, listing mutations and deletions in the RamAR system that had not previously been described.The wide diversity of mutations identified suggests that clinical strains do not have a specific effluxregulating response to antibiotics.Several evolutionary strategies are associated with RamR in MDR strains to reduce its repressive action, making RamA a key efflux regulator in the Klebsiella-Enterobacter-Salomonella group.Specific remarks : Introduction section: -Line 77: ramAR has also been studied in K. aerogenes (Chollet et al. Antimicrob. Agents Chemother. 2004).Please note that ramAR does not exist in E. coli (mentioned delayed in the discussion).

Results section :
-Line 143 and legend of tables : Phe-Arg-ß-naphthylamide (pAßN) to be replaced by PAßN -Line 194: Interestingly, this deletion 1_69 was already observed in E. cloacae in a recent study (Ferrand A. et al., J Antimicrob Chemother. 2023) Discussion section: -Line 250: The genes of activators such as MarA, SoxS, RamA, etc. are not mutated, as these global regulators regulate various physiological processes and their modification would have a deleterious impact on the bacterium.Nevertheless, mutations have been regularly described in the repressors MarR (especially in E. coli), SoxR in various species and Rob.-Line 277 : amino acid key position 154-155 was also of concern in K. aerogenes (Ref 19) and associated to resistance to chloramphenicol.
Reviewer #2 (Comments for the Author): Gravey, Michel et al. examined ESBL-producing isolates collected from intensive care units for the so-called t2c2 phenotype (resistance to temocillin, tigecycline, ciprofloxacin and chlo-ramphenicol), which is due to mutations within the ramAR locus.The t2c2 phenotype was assessed using standard MIC assays and genome comparisons were based on Illumina se-quencing datasets.The authors observed the t2c2 phenotype in 7% of all ESBL-E isolates analyzed, including 16 K.pneumoniae strains and 15 Enterobacter cloacae complex strains.Due to the reversibility of the t2c2 phenotype by the efflux pump inhibitor Phe-Arg-βnaphthylamide, the authors hy-pothesize a dominant role of RND pumps in the resistance pattern.In fact, the authors found various mutations in ramR, the intergenic ramAR region and acrR, which probably lead to a reduced suppression of acrAB expression and thus to an increased expression of the efflux pump.
The correlation shown by the authors between the t2c2 phenotype and the observed resistance patterns is interesting.Nevertheless, additional experiments should be used to support the authors' core statements.The comments can be found in the review document.
Specific Comments concerning the manuscript intitled : « Central rôle of the RamAR locus in the multidrug resistance in ESBL Enterobacterales ».Spectrum03548-23

General comments :
The authors screened 443 ESBL Enterobacteria for efflux resistance phenotype to 4 antibiotics (temocillin, tigecycline, ciprofloxacin, chloramphenicol), and identified those with mutations in the ramAR locus.The RamA regulatory protein is known to be primarily involved in regulating the active efflux of antibiotics in Klebsiella, Salmonella and Enterobacter, by modulating expression of AcrAB-TolC pump.In this study, the combined efflux-dependent resistance phenotype for the four antibiotics was found only in K. pneumoniae and E. cloacae complex, but not in E. coli.Among these 2 groups, analysis of the sequences of the genes encoding all the regulatory proteins involved in efflux pump regulation, as well as the structural genes of the pump proteins, shows that most of the mutations found in MDR strains are identified at the level of ramR gene and the ramAR intergenic region.This is not a new finding in these bacteria, as previous studies have shown that RamAR plays a major role in the overexpression of AcrAB-TolC efflux pumps, and mutations in RamR have also been identified.However, none of the previous studies analyzed such a large number of isolates and identified so many different mutations at ramAR locus.This work reinforces the observations of a number of previous publications, and has the advantage of having performed a sequence analysis of recently isolated clinical strains, listing mutations and deletions in the RamAR system that had not previously been described.The wide diversity of mutations identified suggests that clinical strains do not have a specific efflux-regulating response to antibiotics.Several evolutionary strategies are associated with RamR in MDR strains to reduce its repressive action, making RamA a key efflux regulator in the Klebsiella-Enterobacter-Salomonella group.

Specific remarks :
Introduction section: -Line 77: ramAR has also been studied in K. aerogenes (Chollet et al. Antimicrob. Agents Chemother. 2004).Please note that ramAR does not exist in E. coli (mentioned delayed in the discussion).

Results section :
-Line 143 and legend of tables : Phe-Arg-ß-naphthylamide (pAßN) to be replaced by PAßN -Line 194: Interestingly, this deletion 1_69 was already observed in E. cloacae in a recent study (Ferrand A. et al., J Antimicrob Chemother. 2023) Discussion section: -Line 250: The genes of activators such as MarA, SoxS, RamA, etc. are not mutated, as these global regulators regulate various physiological processes and their modification would have a deleterious impact on the bacterium.Nevertheless, mutations have been regularly described in the repressors MarR (especially in E. coli), SoxR in various species and Rob.
-Line 277 : amino acid key position 154-155 was also of concern in K. aerogenes (Ref 19) and associated to resistance to chloramphenicol.

Central role of the ramAR locus in the multidrug resistance in ESBL Enterobacterales
Gravey, Michel et al. examined ESBL-producing isolates collected from intensive care units for the so-called t2c2 phenotype (resistance to temocillin, tigecycline, ciprofloxacin and chloramphenicol), which is due to mutations within the ramAR locus.The t2c2 phenotype was assessed using standard MIC assays and genome comparisons were based on Illumina sequencing datasets.
The authors observed the t2c2 phenotype in 7% of all ESBL-E isolates analyzed, including 16 K.pneumoniae strains and 15 Enterobacter cloacae complex strains.Due to the reversibility of the t2c2 phenotype by the efflux pump inhibitor Phe-Arg-β-naphthylamide, the authors hypothesize a dominant role of RND pumps in the resistance pattern.In fact, the authors found various mutations in ramR, the intergenic ramAR region and acrR, which probably lead to a reduced suppression of acrAB expression and thus to an increased expression of the efflux pump.

Major comments
Lines 132-134: The authors mention that 16 K.pneumoniae strains and 15 Ecc strains had the t2c2 phenotype.These strains and their MIC values for TIG, CIP and CHL are also listed in Table 1.However, in the supplementary material (Table 2 and Table 3), the MIC test results are only listed for 11 ECC strains and 9 KP strains.Furthermore, the numbers are not consistent.If one compares the values for e.g.Kp20190611 in Table 1 and Table S2, it can be seen that the values for the inhibitor and the manual measurement are missing in the S2 table and that the MIC for CIP is 8 instead of 64 and for TIG 1 instead of 2.
Lines 143-147: The authors determined the MIC values manually and automatically and found differences in the size of 1 or 2 dilution steps.The treatment with the inhibitor is expressed in the text as "8-fold" etc., but it would be clearer for the reader to express it in dilution steps.A 2-fold reduction means a difference in one dilution step, which could be interpreted as normal variation.In addition, inhibitor treatment lowered the MICs, but some of the MIC values are still high and would be classified as resistant.Did the authors try different inhibitor concentrations?If not, this might be worth a try.
Lines 147-149: The authors should also repeat inhibitor tests for TEM.They argue that TEM MIC values are not affected by the inhibitor.However, this would be a great control to show that the inhibitor has no negative side effects on bacterial physiology, etc.
Lines 168-227: The authors explain in detail the mutations observed in the gene regions of interest, but there is no evidence that acrAB expression and thus the number of efflux pumps is increased in the selected isolates.In other publications in which the authors were involved (https://doi.org/10.1128/aac.00358-23),qPCR experiments were carried out to detect effects on expression, which would also be very helpful for this publication.
Lines 236-243: The authors point out the risk that the t2c2 phenotype is not recognized in diagnostic laboratories.However, CHL and TIG may not be the first-line agents for the treatment of Enterobacterales infections in Europe.Therefore, the significance of t2c2 detection may not be clear to the reader and should be explained in more detail.

Minor comments
An improvement of the language in general is recommended.The high number of spelling mistakes must be corrected.
Lines 155-156: Please reword, it should be made clear that this is not surprising information.
Line 190: Please avoid the wording "behavior of the protein".
Line 574: The authors should improve the resolution of their figures.The labeling of the axes must be larger.
Line 587, 597: The authors should improve the resolution of their figures.

Reviewer 1:
Remark 1: Line 77: ramAR has also been studied in K. aerogenes (Chollet et al. Antimicrob. Agents Chemother. 2004).Please note that ramAR does not exist in E. coli (mentioned delayed in the discussion) Thank you very much for this precision, Klebsiella aerogenes has been added among the list of the species described in introduction with the appropriate reference.The fact that the locus ramAR locus does not exist among Escherichia coli is now clarified.The new version of the manuscript is : Line 74-77: "ramAR locus, which doesn't exist in Escherichia coli, has been described widely among several species like Salmonella enterica serovar Typhimurium (7), Enterobacter cloacae complex (Ecc) (4), Klebsiella pneumoniae (8), and Klebsiella aerogenes whereas marRAB has been well studied in E. coli (9)".
Thank you very much for this remark, the abbreviation pAßN have been correctly replace by PAßN as requested.Line 145 of the revised manuscript: "The addition of 20 mg/L of Phe-Arg-ß-naphthylamide (PAßN) to the medium significantly decreased MICs of CHL, CIP and TIG (Table 1)".Changed were also performed along the manuscript as well as in the Table I title and within the abbreviation's descriptions.
Remark 3: Line 194: Interestingly, this deletion 1_69 was already observed in E. cloacae in a recent study (Ferrand A. et al., J Antimicrob Chemother. 2023).
Thank you very much for this information.The appropriate reference has been added to the article and allow the support of the sentence in the discussion Lines 265 -267 "First, the DNA-binding site could be altered by (i) massive deletions, as it has been found in Ecc strains in other studies (4, 20, 21), and (ii) amino acid substitutions ».
Remark 4: Line 250: The genes of activators such as MarA, SoxS, RamA, etc. are not mutated, as these global regulators regulate various physiological processes and their modification would have a deleterious impact on the bacterium.Nevertheless, mutations have been regularly described in the repressors MarR (especially in E. coli), SoxR in various species and Rob.
Thank you very much for this precision.The discussion part has been rewrite has followed: Lines 247-257: "In both species/complexes, sequences of CsrA, MarA, SoxS, SoxR, RamA, and MarR were conserved, whereas AcrR, ramAR IR and RamR showed the highest proportion of mutations.Consequently, csrA, marA, soxS, soxR, ramA, and marR could be considered as "stable" genes, probably because of their impact on several crucial cellular processes Indeed, CsrA plays a major role in intracellular carbon metabolism by controlling several essential enzymes involved in glucose metabolism in E. coli (17).marRAB and soxRS operons are both involved in oxidative stress responses (10,18).It has been demonstrated that for clinical E. coli strains, the selection pressure constrains the mutations within the marR gene due to important fitness cost (19).Finally, mutations within the soxRS operon have been described among E. coli and K. pneumoniae strains ; they lead to cross antimicrobial resistance phenotype (20, 21) ».
Three new references, associated to this paragraph, have been added in the revised manuscript: Remark 5: Line 277: amino acid key position 154-155 was also of concern in K. aerogenes (Ref 19) and associated to resistance to chloramphenicol Thank you very much for your report which supports our data.In the revised manuscript it is specify Lines 181 -186 "These last have been described as a key positions for protein-substrate interactions in Salmonella enterica serovar Typhimurium (12).Amino acid replacement inside the 155 position or near of it (154) could change the behaviour of the protein, which will result in a lower functionality.Furthermore, deletions within the positions 154 and 155 of the RamR of a K. aerogenes strain was associated with high resistance to chloramphenicol (23) ».

Reviewer 2:
Remark 1: Lines 132-134: The authors mention that 16 K.pneumoniae strains and 15 Ecc strains had the t2c2 phenotype.These strains and their MIC values for TIG, CIP and CHL are also listed in Table 1.However, in the supplementary material (Table 2 and Table 3), the MIC test results are only listed for 11 ECC strains and 9 KP strains.Furthermore, the numbers are not consistent.If one compares the values for e.g.Kp20190611 in Table 1 and Table S2, it can be seen that the values for the inhibitor and the manual measurement are missing in the S2 table and that the MIC for CIP is 8 instead of 64 and for TIG 1 instead of 2.
Thank you very much your remark and taking the time to explore supplementary tables.Indeed, results of manual MICs were missing into the supplementary tables two and three.All the data have been carefully added into the appropriate columns into supplementary tables two and three.This process allows to correct one mistake which was into the table I for the MIC of chloramphenicol for the strain Kp20191103.Importantly, MICs presented into the Table I are those obtained manually.According to the limited ranges of the MICs from the sensititre, it could be a variation between MICs according to the method used.
Remark 2: Lines 143-147: The authors determined the MIC values manually and automatically and found differences in the size of 1 or 2 dilution steps.The treatment with the inhibitor is expressed in the text as "8-fold" etc., but it would be clearer for the reader to express it in dilution steps.A 2-fold reduction means a difference in one dilution step, which could be interpreted as normal variation.In addition, inhibitor treatment lowered the MICs, but some of the MIC values are still high and would be classified as resistant.Did the authors try different inhibitor concentrations?If not, this might be worth a try.
Thank you very much for your remarks.Replacement between fold change and dilutions have been made in the text.In the revised manuscript, results are now presented has follow: lines 146-148 "CHL showed the greatest MIC reduction, between 3and 4-dilutions, followed by CIP (between 0-and 4-dilutions) and TIG (between 1-and 3-dilution) (Table 1)." It is true that inhibitor treatment lowered the MICs and sometimes drugs were still classified as resistant.In our opinion, the limitation effect of PAßN is the result of complex antimicrobial resistance phenotypes.Indeed, excepted the Ecc20200803 strain, all strains had additional fluoroquinolones resistance mechanisms of which the PAßN is inefficient.Regarding chloramphenicol, excepted the strains which add cat of floR genes, all the strains were classified sensible to this antibiotic with 20 mg/L of PAßN.It is true that, for tigecycline, some measured MICs with 20 mg/L of PAßN were equal to 1 mg/L, nevertheless, in all the studied strains, there was a MICs decreased between one and three dilutions.For the strains with a remining MICs at 1 ml/L, it could be hypothesised that, RND pump dysregulation was not the only mechanism involved in the resistance observed.It is true that more important concentrations could be interesting regarding tigecycline resistance, but we didn't try.
Remark 3: Lines 147-149: The authors should also repeat inhibitor tests for TEM.They argue that TEM MIC values are not affected by the inhibitor.
However, this would be a great control to show that the inhibitor has no negative side effects on bacterial physiology, etc.
Thank you very much.As mentioned above, PAßN concentrations chosen have previously been used in several studied without any consequences regarding bacterial physiology.About the TEM MICs in presence of PAßN, interpretation of β-lactams MICs in the presence of PAβN could be difficult as the PAβN has an effect of porins expression OmpC and OmpF.As the consequence an elevation of β-lactams MICs can be observed with PAβN:  for temocillin as mention is our previous article "ramR deletion in an Enterobacter hormaechei isolate as a consequence of therapeutic failure of key antibiotics in a long-term hospitalized patient.Antimicrobial agents and chemotherapy, 64(10), 10-1128"  or for carbapenems which was reported in this article "The efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN) increases resistance to carbapenems in Chilean clinical isolates of KPC-producing Klebsiella pneumoniae.Journal of Global Antimicrobial Resistance, 12, 73-

76."
Remark 4: Lines 168-227: The authors explain in detail the mutations observed in the gene regions of interest, but there is no evidence that acrAB expression and thus the number of efflux pumps is increased in the selected isolates.In other publications in which the authors were involved (https://doi.org/10.1128/aac.00358-23),qPCR experiments were carried out to detect effects on expression, which would also be very helpful for this publication.
Thank you very much, you were completely right.To eliminate this limitation, RNA extractions and qRT-PCR were performed on each mutation found within the ramAR intergenic region and in the RamR sequence.Results are presented in the Table 3 and the results section has been change regarding the new results.
Remark 5: Lines 236-243: The authors point out the risk that the t2c2 phenotype is not recognized in diagnostic laboratories.However, CHL and TIG may not be the first-line agents for the treatment of Enterobacterales infections in Europe.Therefore, the significance of t2c2 detection may not be clear to the reader and should be explained in more detail.
We are right with the reviewer's comment.It is not necessary to highlight the under-estimation of the t2c2 phenotype due to different panel and/or categorization of antimicrobial susceptibility testing among countries.As it represents a minor comment, we prefer to delete this point in our Manuscript Minor comments: Remark 6: An improvement of the language in general is recommended.The high number of spelling mistakes must be corrected.
We are sorry about the presence of spelling mistakes; the manuscript has been carefully corrected.
Remark 7: Line 155-156 Please reword, it should be made clear that this is not surprising information.
Thank you very much, the sentence has been rewritten, and is now: Line 157-158 ".As expected, the ramAR operon was absent from the 194 genomes of E. coli studied."Remark 8: Line 190: Please avoid the wording "behavior of the protein".
Thank you very much, the expression "behaviour of protein" has been removed from the revised manuscript.Thank you very much for these remarks, labelling of the axes has been improved for the figure 1 and resolution of all the figures was increased according to the author's guidelines.
1st Revision -Editorial Decision Re: Spectrum03548-23R1 (Central role of the ramAR locus in the multidrug resistance in ESBL Enterobacterales) Dear Dr. Francois Gravey: Thank you for revising your manuscript.I have received reviews (attached) and another round of revisions is necessary before your manuscript can be reconsidered for publication.While Reviewer 1 was satisfied with the changes, Reviewer 2 has indicated the need for additional editing and has expressed concerns about the presentation of the manuscript.Please ensure that you proofread the manuscript carefully before submitting it again and address all of the comments noted below.
Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, notify me immediately so that the manuscript may be formally withdrawn from consideration by Spectrum.

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To submit your modified manuscript, log into the submission site at https://spectrum.msubmit.net/cgi-bin/main.plex.Go to Author Tasks and click the appropriate manuscript title to begin.The information you entered when you first submitted the paper will be displayed; update this as necessary.Note the following requirements: • Upload point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER • Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file • Upload a clean .DOC/.DOCX version of the revised manuscript and remove the previous version • Each figure must be uploaded as a separate, editable, high-resolution file (TIFF or EPS preferred), and any multipanel figures must be assembled into one file • Any supplemental material intended for posting by ASM should be uploaded separate from the main manuscript; you can combine all supplemental material into one file (preferred) or split it into a maximum of 10 files, with all associated legends included For complete guidelines on revision requirements, see our Submission and Review Process webpage.Submission of a paper that does not conform to guidelines may delay acceptance of your manuscript.
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Sincerely, Shannon Manning Editor Microbiology Spectrum
Reviewer #2 (Comments for the Author): Dear authors, Thank you for processing my notes and comments.In my opinion, the language and the resolution of an illustration are still not good enough and should be revised again.I would like to provide you with the new notes below as a rough guide.
Line 71: "associated with" was correct, please change it again.Line 81: I think "organized in" would be correct.Line 125: Better: "These data show a great diversity of bacterial populations and resistomes among the strains analyzed."Line 138: resistant Line 150: dilutions Lines 157-160: better: The sequences of twelve genes linked to the expression of RND pumps (ramA, ramR, acrR, acrA, acrB, tolC, soxR, soxS, marA, marB, marR, and csrA and the ramAR intergenic region (IR)) were found among the 122 K. 160 pneumoniae and 127 Ecc strains.Line 169: within a regulatory pathway Line 186: fold change Line 245: massive Line 249-251: better: It is noteworthy that some genomic alterations led to a stronger dysregulation of ramA, acrA and tolC than others, suggesting an unequal consequence on the integrity and functionality of RamR.Line 449-452: Better: The expression ratios of ramA, acrA and tolC in strains with a t2c2 phenotype were determined by comparison with the transcription levels of the K. pneumoniae wildtype strain XXX.Please mention which kind of WT strain you have used!Line 632 (and in general): Please write out numbers up to twelve as a word if they are not followed by a unit.Line 630: Figure 1 is still too blurred; you will need to create the image in a different program or increase the resolution when exporting.Line 654-655: Please change this part: "no significative shit".Line 656-657: Correct the sentence.Line 683: Please provide standard deviations.

Line 574 :
The authors should improve the resolution of their figures.The labelling of the axes must be larger.& Line 587, 597: The authors should improve the resolution of their figures.